The process of cellular aging in higher vertebrates is almost invariably accompanied by the massive proliferation of a class of cytoplasmic filaments known as intermediate filaments. Concomitantly with their proliferation during cellular maturation and aging, these filaments become highly insoluble in physiological buffers presumably by increased crosslinking of the filaments to each other. This has been suggested to be one of the main manifestations of the neurons in patients with senile dementia (Alzheimer's disease) but also appears to occur normally during aging. The molecular basis of this process is poorly understood. Here we demonstrate that avian erythrocytes is an ideal system where this process can be dissected biochemically and immunologically during maturation and aging of these cells in experiments which have begun to elucidate the structure, and function of intermediate filaments in these cells. Using a newly developed technique, we have recently shown that intermediate filaments in these cells are composed of two polypeptides, vimentin as their core subunit and a high molecular weight protein, termed synemin, as an accessory protein. Synemin is periodically spaced along the filaments and through self-interaction crosslinks the filaments into a three-dimensional network that interlinks the nucleus and plasma membrane. The degree of crosslinking increases dramatically during erythrocyte maturation and aging. To elucidate the regulation of crosslinking, we plan to characterize synemin from these cells and define the domains on this molecule responsible for self-interaction and the effect of phosphorylation and reduced-oxidized state on this process and on the binding to vimentin filaments. In addition we plan to investigate the localization, structure and enzymatic activity during erythrocyte maturation and aging of a widespread cytoplasmic Ca2+ activated neutral proteinase that exhibits a preferred specificity for vimentin and synemin. The activity of this proteinase towards vimentin and synemin appears to be high during early erythrocyte differentiation but is reduced during erythrocyte maturation and aging. We also plan to purify an inhibitor of the proteinase activity which may be one of the key regulatory elements in allowing increased filament crosslinking during erythrocyte maturation and aging by inhibiting the degradation of synemin. In conjunction with studies on a similar phenomenon observed in other classes of intermediate filaments, in particular neurofilaments and keratin filaments, the studies on the regulation of synemin mediated vimentin filament crosslinking should provide a paradigm and a molecular understanding of the structure and abnormal expression of intermediate filaments during cellular aging and the effect of Ca2+ on this process.